In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal non-catalytic domain, a region that is critical for both subcellular localization and protein-protein interaction. At a semi-permissive temperature, one of these mutants (cdc5-10) exhibited a defective spindle morphology that was not remedied by the provision of CDC14TAB6-1, an allele that allows bypass of both FEAR (Cdc Fourteen Early Anaphase Release) and mitotic exit network. Consistent with the FEAR and the MEN-independent spindle defect, the cdc5-10 mutant arrested in S phase with hydroxyurea treatment possessed significantly shorter spindle length (0.95 ?m) than the isogenic wild-type (1.15 ?m), and exhibited a delayed microtubule nucleation in cells released from nocodazole treatment. Provision of a low-dose of benomyl suppressed the growth defect associated with the cdc5-10 mutation. Consistent with these observations, we found several microtubule binding proteins as putative Cdc5 substrates. For instance, Stu2, a microtubule-associating protein that is important for microtubule dynamics and anaphase spindle elongation, was not properly phosphorylated in cdc5-10 mutant in vivo. Purified recombinant Cdc5, but not the kinase-inactive cdc5(N209A), efficiently phosphorylated purified Stu2 in vitro. Simlilarly, phosphorylation of Slk19, Nud1, and Spc72 were also regulated by Cdc5. These data suggest that Cdc5 may regulate spindle function by directly phosphorylating these proteins. Whether Stu2, Slk19, Nud1, and Spc72 are physiological substrates of Cdc5 and whether the Cdc5-dependent phosphorylation is important for the function of these proteins remain to be further investigated.